Efficacy Evaluation and Optimization of Enzymatic Preparation Process of Anti-platelet Collagen Peptides from Fish Skin

In order to develop a novel functional food with Gly-Pro-Arg(GPR),Gly-Pro-Arg-Gly(GPRG)and Gly-Pro-Arg-Gly-Pro(GPRGP)as the active ingredient,the raw material silver carp(Hypophthalmichthys molitrix)skin hydrolyzed by enzyme that could prevent thrombosis. Firstly,the efficacies of anti-platelet activity of three peptides were evaluated by turbidimetry. It was found that three peptides inhibited ADP-induced platelet aggregation as a dose-dependent manner. Then,the collagen of silver carp skin was hydrolyzed by compound enzyme. Contents of the three anti-platelet peptides in the hydrolysate as the response value was measured. The optimum enzymatic conditions were successively determined by Plackett-Burman test,steepest ascent and Box-Behnken test. Finally,the optimal enzymatic conditions were determined as followed:The substrate concentration of 7%,initial pH8,enzyme concentration of 1930 U/g and hydrolysis time of 8 h. The anti-platelet peptides content(the sum of GPR,GPRG and GPRGP)of the final products obtained under this condition was 6.80%. The IC₅₀ of inhibiting platelet aggregation was 1.669 mg/mL. The physicochemical indexes of the final products were as follow:The total nitrogen content of the product was 17.430±0.065%(on a dry basis),the peptide content was 92.16%±0.27%(based on a dry basis),the hydroxyproline content was 6.42%±0.03%,the peptide(1000 Da)was 94.95%,the ash was 3.92%,and the loss on drying was 0.93%. These indexes of the final products were in compliance with the relevant standards. In summary,an anti-platelet functional food with GPR,GPRG and GPRGP as the active ingredients could be obtained by using the method of this study.